Final Report Summary - NEUROGRAFT (Development of Functionalised Cell Seeded Bioartificial Organ for Transplantation in Nerve Repair) Executive Summary:4.1.1 An Executive Summary The Neurograft collaborative consortium proposed a novel conduit system to aid functional regeneration following spinal cord injury (SCI). The Neurograft collagen-based conduit was functionalised by adding stem cells to further enhance the regenerative potential of neurons guided through the conduit. Extensive in vitro testing led to a conduit prototype which was used in a pre-clinical rat spinal cord injury study. VORNIA LTD:The Neurograft conduit was successfully designed by Vornia from a rat-scale concept that was taken to a human-scale device, developed in an ISO13485 controlled environment and produced under GMP conditions in a process that is translatable to large-scale production. Several prototypes were designed but while the conduit’s compressive modulus and channel design satisfied the requirements, the crosslinking was not able to prevent rapid degradation in vitro and in vivo. However, the key principles for collagen spinal cord conduits are observed and a final conduit model could be fabricated and the deliverables are achieved.NUIG:NUIG provided Vornia with all necessary product specification information to manufacture the Neurograft conduit with all accompanying design and production processes. The original proposition was to further functionalise the conduit by adding an anti-inflammatory cytokine, IL-37. The research carried out by NUIG showed that the Neurograft collagen conduit had the capability to incorporate and release a bioactive anti-inflammatory component; however, an anti-inflammatory effect could not be proven. Thus, extensive testing led to the newly established cytokine IL-10 but, due to the above mentioned delay in conduit production and limited numbers of available new-model conduits, IL-10 could not been tested either in vitro or in vivo during the lifetime of the project. However, experiments with IL-10 will continue at NUIG and the outcome of this promising cytokine as a very promising tool for SCI treatment could lead to further publications after the Neurograft project has finished.STEMMATTERS:Very promising results have been achieved during the initial stages of the project concerning stem cell viability and growth within the IL-37 loaded multichannel conduit, as well as apparent differentiation of these cells towards cells of the neuronal lineage. Later, differentiated cells were further tested for functionality in vitro, by determination of voltage-activated currents, demonstrating the presence of electrically active cells. The combination of cells and conduit, resulting in a combined ATMP, was successfully produced and implanted in a rat SCI model for in vivo functional studies. Invaluable knowledge was generated which allows continuing and more effective pursuit of SCI treatment, expandable to injuries at distinct levels, stages of development and/or aetiologies.NAMSA:Based on clinician’s advice, an implantation in a rat paraplegic model was selected for the first performance while a single transection directly followed by conduit without/with stem cells implantation was chosen for the second study. NAMSA had to overcome many technical obstacles during the experimental phase of the project but was able ultimately, despite huge time pressure, to deliver the requested tasks. Although the histological evaluation is not yet finished, we are very pleased with the outcome of our part of the project since all surgical, behavioural and histological procedures involved in this project are evaluated and established at NAMSA. Moreover, preliminary biocompatibility of the NEUROGRAFT conduit was validated for subsequent file submission to Notified Bodies.OBELIS:Obelis provided a detailed regulatory strategy for the development of each conduit iteration (conduit alone, with cytokine and with cells) to maximise the future relevance of the work for commercialisation or out-licensing of the technologies developed during the project. Throughout all phases of the project Obelis ensured that relevant legislation and standards were followed during planning, designing, manufacturing, packaging and handling of the conduit. Obelis provided extensive training to the other partners on design and development of medical devices and advanced therapy medicinal products.Project Context and Objectives:4.1.2 A Summary Description of Project Context and ObjectivesThe work programme was divided into five work packages, which are briefly described below:Work Package 1: Design and Production of the Collagen-based Neurograft Conduit. The National University of Ireland Galway (NUIG) provided specifications for the prototype and Vornia Ltd designed, fabricated and tested the final conduit to meet the specifications. The final product was packaged, sterilized and used for in vitro and in vivo experiments.Work Package 2: Characterisation of the Neurograft Conduit in vitro. Several protocols have been developed for simulated in vitro degradation using collagenase enzymes (Vornia Ltd) and in vivo degradation (NAMSA) to identify the persistence of the conduit for the subsequent pre-clinical animal studies. To optimise in vitro characterization of stem cells, Stemmatters have chosen adipose tissue as a source for mesenchymal stem cells (MSC). The European Union Tissue and Cells Directives (EUTCD) were followed, and protocols were developed to manufacture these cells to clinical-grade, using xeno-free conditions, compliant with GMP. An extensive quality control panel was established including numerous performance parameters as well as various safety parameters.Work Package 3: Complete in vivo Studies Following SCI under GLP and GMP. NAMSA performed first an in vivo study with the conduit in a paraplegic rat model. An initial contusion was followed four weeks later by a spinal cord resection with/without subsequent conduit implantation. The motor functions were evaluated after contusion and implantation. The inflammation and regeneration of the spinal cord with/without conduits were investigated eight weeks later after implantation using histopathologic evaluation. A second in vivo evaluation of the performance of the conduit was performed using a non-contusion method where a transection only was performed following an immediate implantation of the conduit seeded with cells or conduit alone. Motor function was assessed during the 12 weeks following implantation until the rats were euthanized for histological analysis. Work Package 4: Scientific Coordination and Liaison with European Commission.Work Package 5: Dissemination of Neurograft Project Results to Facilitate Commercialisation.Vornia Vornia’s role in the Neurograft project is the design and production of the collagen-based Neurograft conduit according to specifications from NUIG and the preparation of prototypes for the proposed experiments. After a series of forms was tested to meet these specifications, the device was produced by the injection moulding method. Star-PEG was chosen as the preferred crosslinker. The fabricated conduits were inspected, packaged, and sterilised. Design and FabricationThe original model designed according to the specifications given by NUIG consisted of macro- and micro-channels. After fabrication of the first prototype, members of the Scientific Advisory Board considered that the channels within the conduit should better line up with the white matter tracts of the spinal cord. This advice was followed promptly and a new model was designed and fabricated. Unfortunately, this new conduit appeared to have a much faster degradation rate in vitro than expected. Simultaneously, an in vivo degradation study performed by NAMSA showed that the conduit did not persist in the body for the desired length of time. Therefore, our Scientific Advisory Board advised that a conduit model considering micro-channels only and no macro-channels be produced. Subsequently, the conduit was re-designed again and the fabrication had been altered again and this included a greater degree of cross-linking to reduce the degradation rate. Vornia modified the specification to include degradation time and developed protocols for simulated in vivo degradation using collagenase enzymes and controls with known in vivo degradation times. It was necessary to balance the degradation profile and the compressive modulus, as increased persistence time in vivo will likely correlate with increased compressive modulus. This would have potentially increased the compressive modulus to a point where the device is incompatible with the soft spinal cord tissue. Thus, a number of models have been designed and fabricated during the lifetime of the project: Design 1 had the most channels, but also the highest incidence of channels connecting with each other due to the fragile nature of the collagen between the channels. Furthermore, much of the outer ring of channels appeared to have collapsed. The mean distances between channels in the inner ring was 146 ± 52 µm, the distance between channels in the outer ring was 503 ± 154 µm and the distance between the inner and outer rings was 106 ± 46 µm. Design 2 had a good balance between channel number and positioning. None of the channels were too close together (inner ring: 361 ± 25 µm; outer ring: 845 ± 330 µm; between inner/outer ring: 193 ± 72 µm), and thus there was minimal incidence of channels merging. There was also a relatively large space in the centre with only micro-channels. Design 3 had the same number of channels as Design 2, but with the inner ring closer to the centre and the inner and outer channels radially oriented in phase (distances between channels in inner ring: 189 ± 76 µm; outer ring: 750 ± 237 µm). Although channel merging was not observed in the SEM images, the minimum distance between the inner and outer ring was observed to be as low as 37 µm (average 190 ± 93 µm). In all cases, the average micro-pore diameter was between 30 and 40 µm. The different designs had different benefits and drawbacks: Design 2 had the smallest micro channels and the largest distance between channels, thereby recommending it as the most robust design. Design 3 had the smallest macro-channels when dry, but as this parameter was not demonstrated to be either advantageous or disadvantageous, it was not recommended as a selection criterion.Degradation in vitro and in vivo.The role of the macro-scale channels had to be considered in the degradation process. The conduits with macro-scale channels had a significantly larger surface area without a significant difference in mass. This resulted in relatively faster degradation, which turned out to be a problem. The in vivo studies by NAMSA indicated that the conduits could be observed after 4 weeks subcutaneous implantation. However, in vitro stem cell seeding studies carried out at Stemmatters revealed an unexpectedly rapid degradation rate. Despite passing the specification in collagenase degradation tests, the final design was found to degrade so rapidly in cell culture that it was impossible to recover intact conduits for implantation. Further in vitro verification in cell culture using a fresh batch of conduits also failed to repeat previous favourable degradation rates in vitro. A combination of factors probably contributed to this rapid degradation rate in cell culture: (i) as a crosslinking chemical, 4-arm star PEG is inherently variable and, as a result, the degree of crosslinking achieved batch-to-batch is also likely to be variable, (ii) the fabrication process was not validated prior to manufacture of in-vivo verification batches, (iii) e-beam radiation significantly affects the integrity of 4-arm star PEG crosslinks in the collagen. The reason for the batch-to-batch variability in the degradation rate still remains elusive and would need further extensive testing. However, ultimately the 4- and 8- week in vivo SCI studies could be completed using verification batches which remained intact for at least 4 weeks in stem cell culture.NUIG The Neurograft project aimed to develop a functionalised cell seeded collagen conduit, incorporating a potent immunomodulatory cytokine (IL-37) to target the initial inflammatory response to increase the survival and therapeutic effect of transplanted mesenchymal cells. An in vivo pilot study showed that subcutaneous delivery of IL-37 to animals subjected to contusion SCI significantly reduced astrocytosis and macrophage activation one week post injury.IL-37 Loading to and Release from the ConduitProtein loading analysis showed a high loading efficiency at the rat-scale (64%) and human-scale (60%) conduits. The maximum IL-37 loading dose was 12 μg per mg of collagen. An in vitro release study showed that approximately 3.5% of total protein content is being released within seven days. Moreover, IL-37 remains bioactive following release from hydrogel as it reduces the expression of TNF-alpha in LPS/INF-gamma activated THP-1 cells. Thus, the protein analysis of IL-37 showed a successful result.In vitro Testing of IL-37The in vitro testing of IL-37 was performed using the THP-1 cell line which was directed to a macrophage-like phenotype by treatment with PMA (phorbol 12-myristate 13-acetate).These cells were then ‘activated’ to be pro-inflammatory in nature by adding LPS (lipopolysaccharide) or INF-gamma. This treatment results in the upregulated expression of many cytokines such as the pro-inflammatory TNF-alpha or IL-1. The purpose of these experiments was to determine whether treatment with IL-37 could reduce the expression of TNF-alpha or IL-1. Therefore, IL-37 was added to inflammation activated cells at various time points post treatment. The supernatant from the cells was then collected and ELISA performed to determine the amount of TNF-alpha or IL-1 secreted. Regrettably, there was no situation where either cytokine was significantly reduced by adding IL-37. A similar experiment was also carried out using RAW macrophages with similar results. Acting on advice from our Scientific Advisory Board, the anti-inflammatory environment was re-examined. Replacement for IL-37Based on literature and the use of Proteome Profiler Antibody Array for the parallel determination of selected rat cytokines and chemokines, we selected three potent candidates for anti-inflammatory treatment, IL-4, IL-10 and IL-13. We decided to test the bioactivity of IL-10 first which clearly showed an anti-inflammatory effect of IL-10 after simultaneous treatment of LPS and IL-10, no further iNOS production could be seen.Using Mixed Glial Cell Cultures from the Rat Spinal Cord.In order to mimic more closely the inflammatory response after spinal cord injury, a primary mixed glial cell culture derived from the rat spinal cord was established first. The inflammatory response of primary glial cells and THP-1 cells (used as control) under LPS/IFN-gamma treatment was assessed by measuring the TNF-alpha production. However, even when treated with varying concentrations of LPS (from 10ng/ml to 1000 ng/ml) for 24 hours, rat spinal cord cells did not show an increased expression of TNF-alpha in the supernatant (using Western Blot and ELISA).Thus, we altered the detection markers for measuring inflammation in the primary cell culture by measuring the changes in Nitrite and iNOS production under LPS/IFN-gamma Treatment and could successfully show an upregulation of both markers after inflammation.Stemmatters Mesenchymal stem cells (MSC), obtained from adipose tissue, were the target cell source to functionalize the neurograft conduit. The European Union Tissue and Cells Directives (EUTCD) were followed, and protocols were developed to manufacture these cells to clinical-grade, using xeno-free conditions, compliant with GMP. An extensive quality control panel was established including performance parameters such as cell viability, cell adherence and proliferation, cell immunophenotype, cell tri-lineage differentiation potential, as well as various safety parameters. For the originally planned co-loading of stem cells and IL-37 in the conduit, very high cell adhesion, viability and proliferation were shown, comparable to those of non-IL-37 loaded conduits, demonstrating no negative impact of this cytokine on these parameters. Furthermore, neuronal differentiation culture and subsequent characterization methods allowed the detection of gene expression and protein synthesis of neuronal lineage markers, respectively by qRT-PCR and immunocytochemistry. The markers selected included: neurofilament (NFL) and ßIII tubulin for neurons, myelin basic protein (MBP) for oligodendrocytes, and glial fibrillary acidic protein (GFAP) as marker for astrocytes. Such markers were detected on cells cultured directly within conduit, as well as on cell culture plastic, demonstrating that, for SCI treatment, cells can be loaded to the conduit at a stem cell state or after undergoing neuronal differentiation.Cells cultured in the conduit were analysed by whole-cell patch clamp, and showed the presence of excitable, electrically active neuron-like cells within a heterogeneous population. During cell culture within conduits, it was observed that cells maintain high viability and proliferation. Additionally, conduits with macro channels presented higher degradation rates when in cell culture, as well as structural heterogeneity along conduit length, determined by micro CT and SEM analysis. It was observed that longitudinal micro channels formed during conduit manufacturing would suffice for cell guidance. A successful ATMP was manufactured and implanted in a SCI rat model (by NAMSA). Regulatory documentation was prepared including details of cell sourcing (according to directive 2004/23/EC), cell characterization (identity, purity, composition, biological activity); manufacturing process and components used to isolate, purify and expand the cells; as well as details regarding cell storage.NAMSAFor development of medical devices, safety and performance demonstration is mandatory. Therefore, a series of in vitro and in vivo tests was conducted prior to the performance in vivo study to avoid any major biocompatibility issues. A preliminary rat subcutaneous implantation evidenced a fast degradation kinetic of the conduit. The manufacturing process was modified until a slower degradation was obtained. The biocompatibility of the conduits was then validated (local tissue effects and degradation, cytotoxicity, genotoxicity, systemic toxicity, irritation, endotoxins) under GLP conditions based on ISO 10993 standard. The Neurograft conduit was within the specification in all cases. The first in vivo study with the conduit was performed in a paraplegic rat model. An initial contusion was followed four weeks later by a spinal cord resection with/without subsequent conduit implantation. The motor functions were evaluated after contusion and implantation using the Basso Beattie and Bresnahan (BBB) score. The inflammation and regeneration of the spinal cord with/without conduits were investigated eight weeks after implantation using histopathologic evaluation (under process). Regarding the recovery of the motor function, no statistically significant difference was observed between the BBB scores of the conduit group and control (no conduit) group.The second in vivo evaluation of the performance of the conduit was carried out using a non-contusion method where a transection only was performed following by implantation of conduits seeded with and without cells respectively. Motor function was assessed during 12 weeks after implantation until the rats were euthanized for histological analysis. The BBB scores showed no statistical significance in the recovery of motor functions between the transection only (control) and conduit alone/conduit+cells implantations, respectively. Due to the late start of the in vivo experiments (conduit degradation in vitro, see above), the histological analysis was delayed. Immunohistochemistry of spinal cord cross sections was done using markers for visualisation of astrocytes (GFAP), neurons (beta-tubulin), microglia and macrophages (Iba1), and blood vessels (factor VIII). Additionally, histological staining for collagen (Masson’s trichrome) of spinal cord cross-sections was performed from different rostro-caudal levels.Obelis Obelis was engaged to ensure regulatory conformance (with current and future requirements) and to outline the necessary steps required. Obelis guided all partners in the type/source of information required for a Technical documentation report in accordance with current Good Manufacturing Practices and the requirements of European legislation. This included advice on manufacturing processes, validation, record keeping and Risk Analysis. Guidance was also given on the justification of scientific approaches taken. Obelis further ensured that deviations were in compliance with current and future regulatory requirements. Lloyd’s Register Quality Assurance Ltd (LRQA) had been selected as a notified body for ISO13485.Project Results:4.1.3 A Description of the Main S&T Results/Foregrounds Work Package 1: Fabrication of Multi-Channelled Nerve Conduit Experimental Model Development and Design ReviewThe process of experimental model design and development has been formalised and recorded in the Design History File (DHF) in terms of model design (dependent and independent variables, observations, outcomes and further recommendations) and design reviews associated with the generations. All design decisions have been recorded and thus the design process has been made fully traceable.Developed Project Trace MatrixA project trace matrix which summarises the regulations relevant to different aspects of the project has been prepared and discussed with Obelis as part of regulatory consultation.Preparation of Design Inputs FileA document summarizing design inputs ranging from composition to bioburden to regulatory status of components has been prepared and updated to reflect the ongoing design and development process. Many of the design inputs were drawn from the Product Specification developed in conjunction with NUIG while others were informed by regulatory requirements and manufacturing limitations.Maintenance of Design History FileThe design history file has been maintained and updated as the design process progresses. Significant focus has been placed on the regulatory requirements for a class III device implanted into the spinal cord (considered to be the intrathecal space). As this is an implantable, degradable device in a high-risk space, the regulations surrounding pyrogens and sterility are particularly demanding. The relevant regulations to this project identified to date are: EN556, ENISO11137, ENISO11607, ISO10993, ISO11737, ISO14644, ISO13908, ISO15223 and ISO22442. Further regulations are likely to be relevant and guidance will be given from Obelis.Notified Body SelectionLloyd's Register Quality Assurance Ltd (LRQA) has been selected as Vornia’s Notified Body for ISO13485 and as a potential approval body for Neurograft CE marking. LRQA has experience in the medical device area including medical devices that include tissues or animal derived products and neural products – a specialty area. LRQA awarded Vornia certification for Design and Development of medical devices after an audit in 2013. A follow-up audit to approve Vornia’s certification for manufacture and supply of medical devices took place in October 2014. This accreditation was maintained following inspection in August 2015.Fabrication:Three different conduit designs have been fabricated during the life time of the project. They are summarised in Table VN-T1.1.1: Summary of all delivered conduits to the partners. Please see attachment.As the device utilizes bovine-derived collagen, the regulatory pathway for the conduit as a medical device must also follow the requirements of Commission Regulation (EU) No 722/2012 of 8th August 2012 concerning particular requirements as regards the requirements laid down in Council Directives 90/385/EEC and 93/42/EEC with respect to active implantable medical devices and medical devices manufactured utilising tissues of animal origin. This Regulation applies to the use of materials derived from TSE-susceptible species in medical devices and sets out specific requirements for the sourcing, testing and processing of materials from these species. It requires a detailed risk analysis to be prepared by the manufacturer for the Notified Body, and requires the Notified Body to consult with their national Competent Authority on the acceptability of using the animal tissue in the medical device. Subsequent consultation by the NCA with all other national CA of the Member States is then undertaken to gain approval for use of the animal-derived material.If the material has a Transmissible Spongiform Encephalopathy (TSE) certificate of suitability issued by the European Directorate for the Quality of Medicines (EDQM), no further information on animal source, procurement, husbandry and slaughter processes and manufacturing method for the material is required by the Notified Body, but the risk assessment and justification for inclusion of the material must be included in the submission to the NB. In addition the detailed standards for viral and TSE safety of animal-derived materials established in harmonised EU standards 22442 Parts 1-3 are applicable and Obelis have advised on their requirements for the project.Work Package 2: Characterisation of Neurograft Conduit in vitro.Measurement of IL-37 Release from the Conduit in vitro Collagen scaffolds supplied by Vornia were successfully loaded with IL-37 by the diffusion method. The loading efficiency was investigated by measuring the concentration of fluorescently-tagged IL-37. The bioactivity of released IL-37 could be confirmed by using activated THP-cells and their expression of pro-inflammatory cytokines which was assessed by ELISA. In vitro Testing of IL-37The in vitro testing of IL-37 was performed using the THP-1 cell line which was directed to a macrophage-like phenotype by treatment with PMA (phorbol 12-myristate 13-acetate).These cells were then ‘activated’ to be pro-inflammatory in nature by adding LPS (lipopolysaccharide) or INF-gamma (interferon gamma). This treatment results in the upregulated expression of many cytokines such as the pro-inflammatory TNFalpha or IL-1. The purpose of these experiments was to determine whether treatment with IL-37 could reduce the expression of TNF-alpha or IL-1. IL-37 was added to inflammation activated cells at various time points post treatment, either from stock solution or after release from the conduit. The supernatant from cells was then collected and ELISA performed to determine the amount of TNF-alpha or IL-1 secreted. There was no instance where either cytokine was significantly reduced by adding IL-37. A similar experiment was also carried out using RAW macrophages with similar results (data not shown). Collagen Hydrogel Testing of IL-37In addition to the in vitro testing, a separate study was performed by NUIG where IL-37 was delivered in a collagen hydrogel system to investigate the efficiency of IL-37 in vivo. For this study, inflammation was examined using Iba-1 as a marker for microglia and macrophages, but no additional reduction in inflammation was observed in this IL-37 treated group. The glial scar, the formation of which may also be considered an inflammatory event, was also examined. Again IL-37 offered no additional benefit.Therefore, extensive testing was undertaken to find another suitable candidate to substitute IL-37. The use of Proteome Profiler Antibody Array revealed several potent candidates for anti-inflammatory treatment and one of them (IL-10) was selected, also based on the literature, for further investigation. The bioactivity was successfully shown by using mixed glial cell cultures (see below).CE marking process for the multi-lumen collagen spinal cord implant with addition of an anti-inflammatory substance The addition of any cytokine, anti-inflammatory compound or pharmacologically active substance to the conduit will require additional approval considerations for the Neurograft device. The combination will also be a Class III medical device under Article 1.4 and Rule 13 of Annex IX: Where a device incorporates, as an integral part, a substance which, if used separately, may be considered to be a medicinal product within the meaning of Article 1 of Directive 2001/83/EC and which is liable to act upon the body with action ancillary to that of the device, that device shall be assessed and authorized in accordance with this Directive. All devices incorporating, as an integral part, a substance which, if used separately, can be considered to be a medicinal product, as defined in Article 1 of Directive 2001/83/EC, and which is liable to act on the human body with action ancillary to that of the devices, are in Class III. Obelis’ regulatory staff and consultants provided expert advice to confirm that the device containing IL-37 (or any other anti-inflammatory) will remain a medical device with a device-like primary mechanism of action within the scope of the overall intended purpose of the product – that the conduit itself should guide and support regeneration of the recipient’s nerve fibres. The inclusion of an anti-inflammatory component is intended to modulate the immediate environment to assist in cellular regeneration, but this is within the context of supporting the conduit’s function, not the primary effect of the product in its entirety.Obelis advised the partners on the regulatory impacts of including a medicinal substance within the medical device. This included advice on the studies necessary to support this product, and also detailed requirements on the medicinal substance itself, including quality and safety information which is required to be of an equivalent standard to that for a stand-alone medicinal product. Obelis provided training of the partners on the additional regulatory approval processes for medical devices containing ancillary medicinal substances under Directive 93/42/EEC Annex I Clause 7.4 specifically the assessment of the quality, safety and usefulness of the medicinal substance by a medicines Competent Authority, to ensure that all additional strategic implications of this development were understood by the team and integrated into the development plan.Using Mixed Glial Cell Cultures from Rat Spinal Cord:In order to mimic more closely the inflammatory response after spinal cord injury, a primary mixed glial cell culture derived from rat spinal cord was established first. The inflammatory response of primary glial cells and THP-1 cells (used as control) under LPS/IFN-gamma treatment was assessed by measuring the TNF-alpha production. However, even when treated with varying concentrations of LPS (from 10ng/ml to 1000 ng/ml) for 24 hours, rat spinal cord cells did not show an increased expression of TNF-alpha in the supernatant (using Western Blot and ELISA). Thus, the detection markers were altered for measuring inflammation in the primary cell culture by measuring the changes in Nitrite and iNOS production under LPS/IFN-gamma treatment and could successfully show an upregulation of both markers after inflammation. Furthermore, the anti-inflammatory effect of IL-10 could be clearly shown since after simultaneous treatment of LPS and IL-10 no further iNOS production could be seen. Further qualification and quantification of the mixed glial cell culture was successfully performed using immunocytochemistry (ICC) and flowcytometry. Analysis of Pore Size Distribution and Interconnectivity of New Conduit Formulations (with and without channels) by Micro Computed Tomography (µCT)Three conduit formulations were provided by Vornia: No channels; macroscopically integrate throughout conduit length.Channels; macroscopically integrate at edges but irregular at middle zone of conduit length.Design 5; macroscopically less compact than “no channel” group but structurally more integrated than “channel” group.For µCT and SEM analysis, ~2mm height discs were cut and structural homogeneity throughout conduct length was assessed by sampling a disc on an extremity as at the centre of the conduit (labelled “top” and “middle” respectively). Micro computed tomography (SkyScan 1072) was first performed on dry conduit discs. It could be shown that the “top” disk consisted of a higher distribution of pore sizes, while higher density of smaller pores was quantified in the middle disk. Porosity was fairly high, and the pore interconnectivity was very satisfactory (>90%).Analysis of Pore Size and Morphology of New Conduit Formulations (with and without Channels) by Scanning Electron Morphology (SEM) The same conduit disks that have been used for µCT analysis were further gold-coated and analysed by SEM (JEOL model JSM-6010LV). Imaging was performed from top view of conduit disk, at 15x and 150x magnification. In the “No Channel” group an intact, round structure was observed in both disks. On “top” disc, pores of different sizes and shapes were visible, yet without an organized alignment. The “Middle” disk was apparently slightly more compact than “top”, also with an apparent higher density of smaller pores.Examination of Stem Cell Viability on New Conduit Formulations in the Presence of IL-37Human scale conduits described in Figure STM24-2.4-1 were used. IL-37 loading protocol was provided by NUIG, and, as recommended, IL-37 was purchased at Novoprotein (Cat#C073) and PBS-T loading buffer purchased at Cell Signaling Technology (Cat#9809).Conduit cut into 2mm height were incubated with 175µL of IL-37 solution at 40µg/mL, overnight at room temperature with agitation. After IL-37 loading, the remaining IL-37 solution was collected and stored. Conduits were briefly washed with PBS, and blot-dried to remove excess fluid, immediately before cell loading.Stem cells, human xeno-free Adipose Stem Cells (XF-hASC), Lot 20202800057, 20202800066 and 20202800030 were thawed in xeno-free cell culture media, centrifuged to remove cryoprotectant, and further suspended in xeno-free cell culture media to yield a 2 million cells/ml suspension. 50µl of cell suspension was loaded per conduit disc and incubated 37ºC for 30min before addition of further cell culture media. In vitro Assessment of hASC ViabilityAfter 24h incubation, three and seven days of culture, cell-loaded conduits were assessed for cell viability by staining with live/dead assay, where calcein AM stain live cells green, and propidium iodide stain dead cells red. Cell adhesion was further assessed by SEM.Additionally, cell proliferation within conduits was determined by DNA quantification using Picogreen reagent. High cell viability and homogenous distribution of cells are visible in all conduits.Adipose Stem Cells Potential to Differentiate into Neural Cells upon Transplantation In vitro Assessment of hASC Neural Differentiation Potential and Differentiation of hASC on Conduit in the Presence of IL-37Cell differentiation into the neuronal lineage was tested on the new conduit formulation “No Channel”. Neural differentiation was evaluated, at end of culture, by gene expression (qRT-PCR) of specific neural markers, such as:beta-III-tubulin: microtubule element expressed exclusively in neurons.Neurofilaments (NEFL): intermediate filaments found in neurons; major component of neuronal cytoskeleton.Myelin Basic Protein (MBP): major constituent of myelin sheath of oligodendrocytes and Schwann cells.All genes were normalized for GAPDH endogenous control. Relative expression ratio was determined by the Livak Method 2-ΔΔCt, using data obtained from STM Expansion Media as a non-treatment group. Neural differentiation was further evaluated, at end of culture, by immunocytochemistry of specific neural markers, such as:beta-III-tubulin: microtubule element expressed exclusively in neurons.Glial fibrillary acidic protein (GFAP): intermediate filament protein specifically expressed in astrocytes.In order to evaluate feasibility of loading pre-differentiated cells into conduit, cells were cultured in monolayer and treated under neural differentiation conditions. After 18 days of neural differentiation on “No Channel” conduit or monolayer, beta-III tubulin was overexpressed relative to undifferentiated cells. When pre-differentiated monolayer cells were further loaded to conduits, beta-III tubulin gene expression was modified after the three days of neural maintenance: cells cultured on “No Channel” conduits seem to have slightly increased expression (also comparable to monolayer cells at same time-point: ML-diff d18+3), while those cultured on “Channel” conduits showed considerably reduced beta-III tubulin gene expression.Evaluation of the New Star-PEG Conduits without Channels:Monitor in vitro Degradation During Cell CultureFour new conduit formulations were provided by Vornia, March 2014. Based on data generated in previous tasks, and described in preceding technical reports, cell differentiation into the neuronal lineage occurred successfully when cultured directly on Neurograft conduits. From this point on, this approach will be the preferred for cell differentiation.The 5 cm long conduit of each formulation, both human and rat scale, were used for in vitro culture to assess degradation. Two assays were performed:Full length conduitsConduit discs (2mm height)Human xeno-free Adipose Stem Cells (XF-hASC), Lot 20202800057, 20202800063 were thawed in xeno-free cell culture media, centrifuged to remove cryoprotectant, and further suspended in media to yield a 2 million cells/ml suspension. Cell suspension was loaded per conduit (50µl /disc; 1250µl/ full length, human scale) and incubated at 37ºC for 30 min before the addition of further cell culture media. These were cultured for three/four days in neural pre-induction media, after which neural differentiation media was used for an additional ten days. As control group, xeno-free expansion media was used for culture.Note: Cell seeding protocol described in previous reports included loading of IL-37 onto conduit before cell seeding. Given data provided by NUIG regarding inefficacy of IL-37, no further loading on conduits was performed during in vitro testing. Determine Porosity, Pore Size, Interconnectivity and Distribution by µCTFor µCT analysis, ~2mm height discs of the dry conduits were cut with a blade. Structural homogeneity throughout conduct length was assessed by sampling a disk on the extremities and at the centre of the conduit (“top”, “middle”, “bottom”). Micro computed tomography (SkyScan 1072) was first performed on dry conduit discs. After in vitro neural differentiation, conduit discs were dried and analysed by micro CT, according to the protocol described above. When comparing frequency of pore size within group B discs before in vitro culture, the middle and bottom disks displayed similar pore size and distribution profile, with higher frequency of smaller pores (around 100 µm), and presence of pores up to 400 µm, while the top disc showed pore sizes up to 500 µm, and higher frequency of pores around 200 µm. Porosity was fairly high throughout the conduit, yet pore interconnectivity was decreased in the middle of the conduit (76%), as compared to both extremities (>90%). This heterogeneous profile throughout the conduit length was consistent with macroscopic observation during in vitro culture.Assess Electrical Activity of in vitro Neuro-Differentiated hASC by Patch-ClampPatch-clamp assay was used to identify and record electrical currents occurring through ion channels, and therefore determine whether the neuro-differentiated cells are excitable cells, such as neurons. Records of single cells, differentiated on Neurograft conduit, were obtained at controlled current and controlled voltage. The recordings revealed both, potassium activated channels, and sodium activated channels were detected. Additionally, it was shown that these neuron-like, electrically active cells are present within the heterogeneous population of cells. Microscopic observation revealed three morphological different cell types: A) neuron-like cells containing one long process and multiple shorter process; B) astrocyte-like cell containing multiple short process; C) interconnecting cells with long and short process.Define Protocol for Delivery of Conduit+Cells for in vivo Main StudyProtocol for delivery of conduit+cells for in vivo implantation was defined based on 2 fundamental criteria: structural integrity of conduit after in vitro neural differentiation of cells on the conduit and electrical activity of cells. Authorisation process for collagen conduit functionalised with neural stem cellsAddition of viable neural stem cells will cause the combined product to be regulated as an advanced therapy medicinal product under the ATMP Regulation 1394/2007. Therefore the combination product will be authorised as a medicinal product and CE marking will not be applicable. However the data generated during the project has been guided and reviewed by Obelis to ensure that it is of appropriate suitability to be used in the future development of such an ATMP.Immunohistochemistry Testing using Paraffin- or Cryo-Sections.Stemmatters (STM) received 7 rat spinal cord explants from pilot in vivo study performed at NAMSA:- 2 explants with contusion, without conduit implantation (animals #3, #5)- 5 explants with contusion, with conduit implantation (animal #1, #2, #4, #7, #8)Samples for paraffin-sectioning underwent standard dehydration with ethanol solutions and paraffin embedding. Samples for cryo-sectioning were included in OCT. Both were included for sagittal and horizontal sectioning. Various cell types and structures could be successfully identified using specific antibodies: GFAP (astrocytes), β-tubulin 3 (neurons), NG2 (Chondroitin sulphate proteoglycan), Neurofilament, Iba1 (microglia and macrophages), factor VIII (vascularization), Masson’s trichrome (collagen). Work Package 3: Complete In vivo Studies Following SCI under GLP and GMPGMP Manufacturing of Neurograft ATMP Samples for in vivo GLP Study #194275Fifteen 10 mm long rat scale conduits (batch 7020RS00x) provided by Vornia were shipped at room temperature (29th May 2015) and received by STM at 09th June 2015. These conduits were used for in vitro culture and neural differentiation but unfortunately were highly fragmented after 14 days upon arrival at NAMSA for the in vivo study and thus, could not be implanted.In vitro Degradation Test of Rat Scale Conduits Shipped 09th June 2015 and 08th July 2015Three 10 mm long rat scale conduits (batch 7020RS00x and 7025RS00x) provided by Vornia were used. Conduits were shipped at room temperature, with gel pack (07th July 2015), received by STM at 08th July 2015 (1 day in transit). Conduits were used for in vitro culture as described in section I, non-cGMP. XF-hASC Lot 20202800063 was used. Conduits were macroscopically and microscopically observed.In vitro Degradation Tests of Rat Scale Conduits Shipped 08th July 2015 and 06th August 2015Three 10 mm long rat scale conduits (batch 9017RS001 and 9017RS002) provided by Vornia were used. Conduits were shipped at room temperature, with gel pack (05th August 2015) and received by STM at 06th August 2015 (1 day in transit). Conduits were used for in vitro culture as described in section I, non-cGMP. XF-hASC Lot 20202800063 was used. Conduits were macroscopically and microscopically observed.Manufacturing of Neurograft ATMP Samples for in vivo Study #204881Six 5 mm long rat scale conduits (batch 3025RS001x) were provided by NAMSA, shipped at -20ºC (14th September 2015) and received by STM at 15th September 2015 (1 day in transit). Conduits were used for in vitro culture as described above. Each conduit was cut in half, resulting in two 2.5 mm long conduits. Conduits were macroscopically and microscopically observed. The twelve 2.5 mm long conduits batch #3 were seeded with hASC, cultured in neural pre-induction media for three days, after which they were supplemented with neural differentiation media. After a total of 15 days of in vitro culture, all conduits remained intact and could be successfully used for implantation.In vivo Pilot StudyImmunohistochemical analysis of seven rat spinal cord explants has been performed. The samples were processed for paraffin and cryosectioning, respectively. Sections were performed and immunohistochemistry protocols executed for the same set of antibodies that had been chosen for the two main studies.Main Spinal Cord Injury Studies Two in vivo studies were conducted. The first experiment (Study number 194275) involved observation of 31 rats after implantation of the Neurograft conduit for up to 8 weeks. The second study (Study number 204881) involved 36 rats followed up to 12 weeks after implantation of the Neurograft conduit.Selection of the Animal ModelThe model, to be used for evaluation of performance of the product to show that the device produces the intended effect relative to the targeted medical conditions, was extensively discussed with the scientific advisory board. The use of the rat model was confirmed. However, to better simulate the clinical condition, the use of a contusion model followed by the transection model in the rat was proposed, instead of the hemi-section initially proposed. The contusion allows simulation of a mechanical trauma to the spinal cord which will set up a cascade of widespread progressive biochemical and cellular processes (also called secondary injury). This model was selected for the first study (n° 194275). This was intended to mimic the clinical situation when a transection injury occurs and implantation of the Neurograft collagen conduit can be considered only when no spontaneous motility recovery is expected. It should be noted that this model is not yet described in the literature that mainly reports acute primary injury. The time periods were also adapted based on the literature and experience of the members of the scientific advisory board. It was agreed to follow-up the animals for up to 12 weeks, which was considered adequate to evaluate the performance of the Neurograft collagen conduit.The design of the second in vivo study (n° 204881) was discussed with the partners and the scientific advisory board. The new design took into account the observations made during the first study 194275. This study involved a primary contusion before transection of the spinal cord and Neurograft conduit implantation. In this newly developed model, the inability to see the injured portion of the spinal cord after the contusion prevented verification of the contact of the Neurograft conduit with healthy spinal cord stubs. For this reason, the contusion was no longer performed. Even if the contusion better approximated to the clinical situation, it added more variability to the results with parameters that could not be controlled. It was considered that an implantation in a healthy transected spinal cord was different from the clinical situation but still adequate and more robust for a proof of concept demonstration.Stemmatters showed that the Neurograft conduit from batch number three were stable. The number of conduits available allowed implantation of only short conduits (2.5 mm long).For these reasons, a single transection of a healthy spinal cord coupled with the implantation of a short conduit was selected to further evaluate the in vivo performance of the Neurograft conduit seeded or not with cells.Successfully Performed First Study - Eight Week Time Period - Study n° 194275The purpose of this first study was to evaluate the performance (regeneration of spinal cord function) of the Neurograft conduit after a spinal cord injury. This study consisted of two different surgeries. Firstly, the spinal cord at thoracic vertebras T8 to T10 was exposed and underwent a moderate contusion at T9 with an impactor device to induce paraplegia of the rat. At four weeks after contusion, the rats with persistent signs of paraplegia underwent a second surgery as follows:Test group: the injured spinal cord was surgically removed and replaced by the Neurograft conduit alone.Sham-operated group: the spinal cord was exposed but left untreated. This group mimicked the clinical situation.Control group: the spinal cord underwent a simple transection at T9 without Neurograft conduit implantation. This group was done to evaluate the effects of the transection itself and allowed comparison with the test group.During eight weeks after implantation, the technical and biological performance of the scaffold (absence of migration, adhesion to the spinal cord, restoration of a physical continuity of the spinal cord, motor function recovery) was assessed using macroscopic observations and motor function evaluation.Figure NAMSA 3.2.1: Study design. Please see attachment. The motor function of the hind limbs of all rats was evaluated at day 3, 7, 14, 21 and 27 after the first surgery. Care was taken so as to minimize rat discomfort and reduce paraplegia-related illnesses (pressure sores, urinary tract infection and hypothermia). The paraplegic rats underwent the second surgery and were allocated to one of the three groups described previously (test, control and sham-operated groups). The absence of difference of the BBB scores obtained before the second surgery among the three groups was verified with an ANOVA test with a 5% alpha-risk. During the second surgery, the spinal cord was transected. A single transversal cut was performed in the control group while a double transversal cut with removal of injured portion of the spinal cord (approximately 6 mm long) was performed in the test group. The defect of spinal cord was implanted with a Neurograft conduit. Ease of use and simplicity of cutting of the Neurograft conduit was assessed by the surgeon. The motor function of the hind limbs of the rats was evaluated weekly up to eight weeks after the first surgery. All animals were euthanized eight weeks after the second surgery and the spinal cord was observed macroscopically. The absence of migration of the Neurograft conduit, adhesion to the spinal cord, and restoration of a physical continuity of the spinal cord were assessed. The spinal cord was sampled and fixed for further evaluation of inflammation and regeneration through the Neurograft conduits by comparison with the sham-operated and control groups.Successfully Performed Second Study – 12 Week Time Period - n° 204881The purpose of this second study was to evaluate the performance (regeneration of spinal cord function) of the Neurograft conduit over a longer period of time.In contrast to the first study, the spinal cord of 36 rats was transected and the created spinal cord gap was bridged immediately with the Neurograft conduit alone (test group 1) or seeded with human neural-differentiated stem cells (test group 2). A group of rats underwent the transection procedure without Neurograft conduit implantation to serve as a reference (control group).During twelve weeks after implantation, the technical and biological performance of the scaffold (absence of migration, adhesion to the spinal cord, restoration of a physical continuity of the spinal cord, motor function recovery) are assessed using macroscopic observations and motor function (ongoing analysis).In contrast to the first study, an immunosuppressant drug (cyclosporine A) was administered to all rats because the Neurograft conduit was functionalized with human neural-differentiated stem cells (test group 2).The motor function of the hind limbs of the rats was evaluated weekly up to 12 weeks after the surgery until they had been euthanized and the spinal cord was observed macroscopically. The absence of migration of the Neurograft conduit, adhesion to the spinal cord, and restoration of a physical continuity of the spinal cord was assessed. The spinal cord was sampled and fixed for further evaluation of inflammation and regeneration through the Neurograft conduits by comparison among groups.Toxicology StudiesThe pre-clinical safety corresponds to the absence of unreasonable risk of illness or injury with the use of the device for its intended uses and condition. This corresponds to the evaluation of the biocompatibility of the Neurograft collagen conduit first, then the combination of the conduit with cells (Advanced Therapeutic Medicinal product). The safety testing was not described in detail in the proposal, and corresponds to the generic term of “toxicology”. The first step of this evaluation was to initially define what was needed based on the Neurograft collagen conduit, its composition and its clinical use. Extensive discussions took place with Vornia and Obelis about the regulatory requirements for this conduit.A first set of biocompatibility tests was performed and were GLP compliant. These tests were selected based on the quantity of conduits available. The biocompatibility tests were performed on the sterile human-scale Neurograft conduit because this device will be used for potential clinical evaluation.These tests were performed following regulatory guidelines applicable for each test, based on the ISO 10993 standard. For each test, a GLP protocol and report was provided.Table NAMSA 3.2.1. Summary of biocompatibility tests and results. Please see attachment.Tissue Effects and Degradation (Study 194277). Several conduits were tested to verify the degradation kinetic of the conduits after subcutaneous implantation. This helped to refine the prototypes in order to get a degradable kinetic compatible with the clinical use (i.e. more than four weeks). When the prototype was selected, a GLP study was conducted. The presence of any adverse local effects in the tissues surrounding each conduit and the article degradation was macroscopically and histopathologically evaluated. MTS Cytotoxicity Test (Study 179119)This in vitro study was to evaluate Neurograft for potential cytotoxic effects following the guidelines of ISO 10993 5, Biological Evaluation of Medical Devices, Part 5: Tests for in vitro Cytotoxicity. A single preparation of the test article was extracted in single strength Eagle Minimum Essential Medium (EMEM10) at 37+/-1°C for 24+/-2 hours. A negative control, reagent control and a positive control were similarly prepared. Following extraction, the test article extract was diluted in EMEM10 to obtain concentrations of 50%, 25% and 12.5% (v/v). The positive control extract was diluted in EMEM10 to obtain concentrations of 25%, 20%, 15%, 10% and 3% (v/v). Triplicate monolayers of L 929 mouse fibroblast cells were dosed with the full strength and diluted extracts and incubated at 37+/-1C (humidified) in presence of 5+/-1% CO2 for 24-26 hours. Following incubation, 20 µl of the MTS-PMS solution, prepared just before use, were dispensed in each well and incubated during 120-135 minutes at 37+/-1°C (humidified) in 5+/-1% CO2. The percent viability for the test article was determined from the reagent control. A decrease in the number of living cells results in a decrease in the metabolic activity in the sample. This decrease directly correlates to the amount of brown formazan formed, as monitored by the optical density at 492 nm. Genotoxicity: Bacterial Reverse Mutation (Study 179120)The test article Neurograft was extracted at 37 ± 1°C for 72 ± 2 hours in 0.9% Sodium Chloride solution (NaCl) and 95% Ethanol (EtOH), using a ratio of 3 cm²/ml. A bacterial reverse mutation assay was conducted to ascertain whether the extracts would induce reverse mutations at the histidine locus of the Salmonella typhimurium tester strains TA98, TA100, TA1535 and TA1537 or at the tryptophan locus of Escherichia coli tester strain WP2uvrA. The assay was conducted in the presence and absence of metabolic activation. This study was based on the requirements of OECD guideline 471 (1997) mentioned in the ISO 10993 part 3 standard (2014).Tubes containing molten top agar were inoculated with culture from one of the five tester strains, along with the test article extracts tested at the single dose of 100µl/plate of the 100% extract. An aliquot of phosphate buffer or rat liver S9 Mixture providing metabolic activation was added. The mixture was poured across triplicate plates. Parallel testing was conducted with negative and positive controls. The mean number of the test extract plates was compared to the mean number of the appropriate negative control plates for each of the five tester strains.ISO Acute Systemic Toxicity in Mice (Study 179121)An acute systemic toxicity test was performed on the Neurograft conduits to determine the systemic toxicity risk induced after injection of the test article extracted in 0.9% NaCl and in sesame oil on mice. This study was conducted according to the requirements of the ISO 10993 standard - Part 11 (2006): Tests for Systemic Toxicity. The extracts were prepared as follows: - Extraction vehicles: 0.9% NaCl and sesame oil- Duration: 72 hours +/- 2 hours under agitation- Ratio test article/vehicle: 3 cm²/ml - thickness >= 0.5 mm - Temperature: 37°C +/- 1°CA single dose of each extract was injected into five mice per extract, by the intraperitoneal route, at the dose of 50 ml/kg. Similarly, five mice were injected with each corresponding extraction vehicle. The animals were observed immediately and at 4, 24, 48 and 72 hours after systemic injection. They were weighed at 24, 48 and 72 hours after injection. Intracutaneous Irritation in Rabbits (Study 179122)An intracutaneous test was performed on the Neurograft conduits to evaluate the potential of the material to produce irritation following intradermal injection of 0.9% NaCl and sesame oil test article extracts in the rabbit. This study was conducted according to the requirements of the ISO 10993 standard: Biological Evaluation of Medical Devices, Part 10 (2010): Tests for Irritation and skin sensitization. The extracts were prepared as follows: - Extraction vehicles: 0.9% NaCl and sesame oil- Duration: 72 hours +/- 2 hours under agitation- Ratio test article/vehicle: 3 cm²/ml - thickness >= 0.5 mm - Temperature: 37°C +/- 1°CThree rabbits received by intracutaneous route on the left side five injections of 0.2 ml of the 0.9 % NaCl extract and five injections of 0.2 ml of the sesame oil extract. Similarly, on the right side, the rabbits received five injections of 0.2 ml of the 0.9 % NaCl extraction vehicle and five injections of 0.2 ml of the sesame oil extraction vehicle. The sites were examined immediately and at 24, 48 and 72 hours after injection, for gross evidence of tissue reaction, such as erythema and oedema.Bacterial Endotoxin Quantification (Study 207200)The test articles were immersed in a non-pyrogenic and sterile vial containing 40 mL of pyrogen-free water. The test articles were then submitted to the following treatment:- vortex agitation for 30 seconds, at room temperature (+ 15°C / + 25°C).- orbital agitation at 200 rpm, for 20 minutes, at room temperature (+ 15°C / + 25°C)- vortex agitation for 30 seconds, at room temperature (+ 15°C / + 25°C).The extracts were tested pure, diluted at 1/10 and 1/100. A standard curve from 5 IU or EU/ml to 5.10-3 IU or EU/ml was prepared. A negative control in LAL free sterile water was performed. An inhibition or enhancement test was conducted in parallel. A known concentration of endotoxin of 0.5 IU or EU/mL was added to two spares well of extract. Bacterial endotoxin rates were determined using spectrophotometric measures at 405 nm.Analysis of Spinal Cord Explants Regarding Inflammation and Regeneration Through the ConduitsSpinal cords were sampled at the end of the in vivo phase of SCI and processed by NMSA for further analysis. First Study - 8 W Time Period - Study n° 194275For each rat, the samples of spinal cord were approximately 20 mm long and included the injured portion of the spinal cord. It was cut into 5 pieces of 4 mm long and transverse sections (approximately 4.5 µm thickness) were prepared using a microtome (Microm®). Approximately 220 slides for each rat were collected.The sections were grouped in sets for immunohistochemistry using antibodies against the following markers:GFAP: Glial Fibrillary Acidic ProteinNF: NeurofilamentIBA1: Ionizing calcium-Binding Adaptor molecule 1 (IBA1)NG2: Chondroitin sulphate proteoglycanAMBP: Anti-Myelin Basic ProteinFactor VIIIThe stained sections were transferred to the National University of Ireland Galway for further analysis.Second Study – 12W Time Period - n° 204881The tissue collection procedure was the same as that for the first study. Although immunohistochemistry could not be performed due to the termination of the project, the collected and embedded spinal cords were sent to NUIG for analysis.Potential Impact:4.1.4 The Potential Impact and the Main Dissemination Activities and Exploitation of Results The successfully designed Neurograft conduit was taken from a rat scale concept to a human scale device. Since it was also developed in an ISO13485 controlled environment and produced under GMP conditions, the conduit would be translatable to large-scale production and thus, fabricable in the future for the treatment of spinal cord injury. The conduit may be loaded with anti-inflammatory agents and stem cells when complete and this combination bio-artificial organ will have a huge impact in the area of spinal cord regeneration as there is no comparable product on the market. In Europe, there are approximately 330,000 people living with spinal cord injuries with more than 11,000 new cases reported annually . The costs to the European healthcare system in maintaining, treating and rehabilitating these patients is over €15.5 billion per year1. Should a European treatment for spinal cord injury become available, it would have significant financial benefit in addition to the benefit to the patient’s quality of life. According to the World Health Organization (WHO - Spinal cord injury, Fact sheet N°384, Nov 2013), nearly 500,000 people suffer a spinal cord injury per year, worldwide. As a consequence of mobility barriers, injured people become dependent on caregivers; 20-30% of patients develop depression which impairs functional and overall health improvements, and secondary health conditions are common2. Altogether, these impairments result in reduced participation in society, where the adult unemployment rate exceeds 60% and child enrolment in school is similarly affected2. Concerning costs to SCI, direct costs are related to level of injury, and indirect costs related to loss of earnings, which often exceed the direct costs. Ultimately, people with SCI have a two to five time greater probability of premature death . A successful therapeutic approach of SCI, where functionality of neuronal tissue is re-established through the use of stem cells delivered within a structural biomaterial, would have an invaluable socioeconomical impact for the millions of people affected directly and indirectly by spinal cord injuries.The socioeconomical impact of NEUROGRAFT can exceed that which relates to clinical improvement of SCI patients. The European medical technology sector will grow and become more competitive through the expertise generated under the project, specifically in the field of regenerative medicine. The consortia’s SMEs became more knowledgeable with respect to manufacturing of medical device(s), selection of therapeutic stem cells, as well as manufacturing of a combined ATMP. Consequently, business in this sector will create new opportunities for employment of highly qualified professionals. Of equal impact is the possibility to expand concepts and technologies developed under NEUROGRAFT to other clinical indications. Peripheral nerve repair might constitute one of the closest to market therapeutic targets, while others might still be explored.Ref.: Parliamentary Assembly, Council of Europe Recommendation 1560. http://www.who.int/mediacentre/factsheets/fs384/en/ WHO - Spinal Cord Injury, Fact sheet N°384, Nov 2013.List of Websites:4.1.5 Project Public Webpagehttp://neurograft.eu/
